Targeted enzymatic therapy for coeliac disease.

Coeliac disease (CD) is an autoimmune enteropathy triggered by proline-rich gluten immunogenic peptides (GIPs), for which no curative therapy exists. We developed celiacase (Clc), a recombinant prolyl endopeptidase engineered from pitcher plant neprosin to enhance expression, stability, and activity. Clc showed maximal activity at gastric pH, synergized with and resisted pepsin, and efficiently cleaved GIPs, including the highly immunogenic 33-mer, outperforming Aspergillus niger prolyl endopeptidase in degrading GIPs from wheat flour and gliadin. At an enzyme-to-gliadin ratio of 1:250, Clc reduced GIP levels by up to 99% in a dynamic human gastrointestinal simulator. Ex vivo, Clc-digested 33-mer fragments failed to elicit cytokine responses in mouse and rat macrophages and duodenal biopsies from CD patients. In vivo, low-dose Clc (1:75-1:380) degraded gliadin and attenuated pathology in gliadin-fed mice, reducing villus atrophy, inflammation, antibody responses, and gluten-induced dysbiosis, while restoring immune-regulatory markers and microbial metabolic pathways. In summary, Clc is a potent, acid-stable glutenase with promise as a therapeutic adjunct or alternative to a gluten-free diet for CD patients.